The detection and quantitation of antigenic substances in biological samples frequently utilize immunoassay techniques. These techniques are based upon the formation of a complex between the antigenic substance being assayed and an antibody or antibodies in which one or the other member of the complex may be detectably labeled. With competitive immunoassay techniques, the antigenic substance in a sample fluid being tested competes with a known quantity of labeled antigen for a limited quantity of antibody binding sites. The amount of labeled antigen bound to the antibody is inversely proportional to the amount of antigen in a sample.
By contrast, most immunometric assays employ a labeled antibody. In such an assay, the amount of labeled antibody associated with the complex is directly proportional to the amount of antigenic substance in a fluid sample.
In sandwich immunometric assays, a quantity of unlabeled antibody is bound to a solid support which is insoluble in the fluid being tested. This immobilized antibody is first contacted with the sample being tested so that a binary antigen-antibody complex is formed. After a suitable incubation period, the solid support is washed to remove unbound antigens, then contacted with a solution containing a known quantity of a second antibody. After a second incubation period, the solid support is then washed a second time to remove the unreacted antibody. A labeled anti-antibody to the second antibody is then added, allowed to incubate for a sufficient amount of time, and the complex then washed. The washed solid support is then tested to detect and quantify the presence of labeled antibody, for example by measuring the emitted radiation of a radioactive label. The amount of labeled antibody detected is compared to that for a negative control sample. This type of assay is frequently referred to as a two-site or sandwich assay, since the antigen has two antibodies bonded to its surface at different locations. Despite their great utility, sandwich immunoassay has been recognized to be a slow procedure, in part because washing steps are required and lengthy incubation periods are required to reach equilibrium. David, et al., U.S. Pat. No. 4,376,110.
To eliminate at least one of the washing steps associated with this procedure, so-called simultaneous and reverse assays have been developed. A simultaneous assay involves a single incubation step as the antibody bound to the solid support and the labeled antibody are both added to the sample being tested at the same time. After incubation, the solid support is washed to remove unbound analyte and unbound antibody, and the bound antibody-analyte-labeled antibody "sandwich" is detected as with a conventional "forward" sandwich assay. A reverse assay involves the stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation. After a second incubation, the solid phase is washed in conventional fashion and the amount of labeled complex is detected as before. U.S. Pat. No. 4,098,876 to Piasio, et al. However, all of these methods suffer from the requirement for two antigen-specific antibodies which are able to recognize separate and distinct epitopes on an antigen. This is a critical limitation which makes the application of such immunoassays impractical for small antigens.
Immunoassays which require only one antigen-specific antibody are preferred. Such an immunoassay was described by Hirano, K. et al., Anal. Biochem. 154:624-631 (1986) who disclose an assay for tumor-specific alkaline phosphatase using a nitrocellulose filter coated with monoclonal antibodies specific for alkaline phosphatase. The presence of the bound analyte was determined by taking advantage of the enzymatic activity of the bound analyte. Thus, this type of immunoassay can only be used for detecting analytes with enzymatic activity which is stable to the conditions of the assay protocol. In addition, such assays are not applicable to detection of all enzymes. For example, enzymes such as thiol protease require the addition of reducing agents to stabilize them. Such reducing agents destroy antibodies by cleaving disulfide bonds.
A dot-blot assay for the detection and quantitation of the Leishmania glycoconjugate was developed which involves dot-blotting the solubilized protein from parasites onto nitrocellulose, blocking the free protein-binding sites with BLOTTO (5% w/v skim-powdered milk) and detecting the presence of the glycoconjugate with an iodinated monoclonal antibody. Handman, E. et al., J. Immunol. Meth. 83:113-123 (1985). A second two-site immunoradiometric assay disclosed by Handman for glycoconjugate involved immobilization of monoclonal antibody on nitrocellulose, blocking remaining protein binding sites with BLOTTO, binding with antigen, followed by a second incubation with the same monoclonal antibody, which was radioiodinated. This assay was based on the fact that Leishmania glycoconjugate possesses a large number of epitopes recognized by the monoclonal antibody. This suggests that Leishmania glycoconjugate contains a repetitive polymeric structure. Handman, supra. Thus, this immunoassay is limited to antigens capable of binding two or more identical antibodies. In addition, assays which rely on radiodinated monoclonal antibody are relatively expensive because of the high costs of monoclonal antibodies and the limited shelf lives of most labeled antibodies.
Another immunoassay which involves a single antibody comprises immobilization of a monoclonal antibody on nitrocellulose, blocking the additional binding sites, and using a labeled antigen to detect the desired antibodies. Suresh, M. R. et al., Anal. Biochem. 151:192-195 (1985). This method is used to screen hybridoma supernatants and to detect monoclonal antibodies. However, in many instances, the antigen is not available in sufficient quantities to allow labeling for use in such an assay. Further, labeling the antigen can result in deleterious alteration of the immuno-specificity of the antigen.
U.S. Pat. No. 4,279,885 to Reese et al., describes a solid phase competitive protein binding assay where an antigen or hapten can be assayed. The method involves competition between the analyte and a labeled form thereof for a limited number of receptor or binding sites which are immobilized to a solid support. The assay may be conducted by mixing the components simultaneously or sequentially. The sequential assay involves contacting a solution of an analyte with a support containing immobilized receptors or antibodies, followed by contacting the mixture with a tracer. The tracer may be the analyte, or analog thereof, which contains a label or tag. Competitive assays are generally recognized to be less preferable to non-competitive assays.
It is also possible to have assays which do not utilize antibodies at all. For example, a sample containing protein to be assayed is mixed with a marker protein in contact with a polystyrene latex. A competition is created between the marker enzyme and the analyte protein for the limited surface binding sites. The inactivation of the enzyme upon binding to the hydrophobic latex surface allows measurement of the bound/free enzyme ratio, and thus, the competing protein concentration. However, this method is not able to distinguish between different proteins, and only gives a measure of the total protein content. Sandwick, et al., Anal. Biochem. 147:210-216 (1985).
Further improvements to immunoassay techniques involve the use of amplification strategies to increase the detection limit. These strategies include substrate cycling and enzyme channeling. Mosbach, K., Ann. N. Y. Acad. Sci. 434:239-248 (1984). However, neither system has been widely adopted.
Thus, it would be desirable to have an immunoassay which is fast and reliable, and requires the preparation of only one specific antibody. Further, it is desirable to have an immunoassay for a multiplicity of analytes utilizing one specific antibody for each analyte to effect detection.